Ultrafast light targeting for high-throughput precise control of neuronal networks

Two-photon, single-cell resolution optogenetics based on holographic light-targeting approaches enables the generation of precise spatiotemporal neuronal activity patterns and thus a broad range of experimental applications, such as high throughput connectivity mapping and probing neural codes for perception. Yet, current holographic approaches limit the resolution for tuning the relative spiking time of distinct cells to a few milliseconds, and the achievable number of targets to 100-200, depending on the working depth. To overcome these limitations and expand the capabilities of single-cell optogenetics, we introduce an ultra-fast sequential light targeting (FLiT) optical configuration based on the rapid switching of a temporally focused beam between holograms at kHz rates. We used FLiT to demonstrate two illumination protocols, termed hybrid- and cyclic-illumination, and achieve sub-millisecond control of sequential neuronal activation and high throughput multicell illumination in vitro (mouse organotypic and acute brain slices) and in vivo (zebrafish larvae and mice), while minimizing light-induced thermal rise. These approaches will be important for experiments that require rapid and precise cell stimulation with defined spatio-temporal activity patterns and optical control of large neuronal ensembles.

In this assumption, the rate equations (1) can be simplified in: For a steady illumination power ./% and an illumination time • ,-, ( the number of tiled holograms and ,-, = 50 µs) the fraction of opsins in O1 state during the illumination is given by and, in the cyclic configuration, the number of transitions from O1 to C1 in the off-time ( − 1) ,-, , is negligeable: In the limit ≪ + , #! ( ) (equation (2)) can be approximated by: and Equation (9) can be written as: 3 Supplementary Note 2:

Cyclic-illumination for multicell excitation: limitations and maximum number of achievable targets
For multitarget excitation, cyclic-illumination using H holograms enables to target = • cells, with m number of cells encoded in each hologram, using √H less total power than conventional holography. Clearly, the gain of cyclic-illumination compared to conventional holography will be higher the larger is the number of holograms H that can be addressed on the SLM and of spots m that can be addressed per hologram. Nevertheless, the values of H and m are submitted to some limitations as discussed in the next paragraphs.

Cell photodamage threshold
For a given non-linear photodamage threshold, Pphd, and a power per cell, Pex, used in conventional illumination, the maximum number of usable holograms Hmax in cyclic-illumination is given by (Pphd / Pex) 2 . Typical illumination conditions used for in vivo 2P optogenetic (conventional parallel illumination at ~200µm depth) have used Pexc corresponding to pulse energy density between 0.06 and 0.5 nJ/µm 2 (corresponding to a pulse energy of 5-60 nJ/cell, a total power of 5-30 mW/cell and a power density of 0.06-0.6mW/µm 2 ) 5-8 at the objective exit, which after correcting for scattering ( .~1 66 µm @1030 nm 9 ) corresponds to ~0.02-0.15 nJ/µm 2 (1.5-18 nJ/cell; 1.5-9 mW/cell ; 0.02-0.09 mW/µm 2 ) at the sample plane. Under our illumination conditions we have found Pphd with a pulse energy density ≈ 1.3 nJ/µm 2 (340 nJ/cell; 170mW/cell; 0.64 mW/µm 2 ) for both FLiT and holographic illumination (Supplementary Figure 17). It follows that hundreds of holograms could be reached before the damage threshold is exceeded so this is not a real limitation to the maximum number of holograms that can be used in FLiT. Limit that is actually mainly imposed by the SLM's properties (pixel number, liquid crystal damage threshold) as discussed in the following section.

SLM pixels per line and damage threshold
As described in the manuscript, cyclic-illumination enables to target = • cells, with m spots generated per each hologram. Increasing the number of H will increase the number of achievable targets but could also limit the number of targets per hologram. Indeed, increasing H requires reducing the vertical size of the tiled holograms and therefore the effective number of pixels per hologram, which in turn imposes a limit to the maximum number of spots m that can be holographically generated with a single hologram.
Additionally, reducing the hologram vertical size requires to correspondingly reduce the size of the illumination beam onto the SLM and therefore increasing the excitation density, PSLM, at the SLM. As a consequence, keeping PSLM below the damage threshold of the liquid crystal SLM also limits the maximum number of spots m per hologram. This is roughly the same value achievable in conventional holography by using a 60 W laser and taking into account the typical power drops mainly due to the TF grating, the overfilling of the SLM screen and objective back aperture, and the limited reflectivity of SLM and mirrors.
To compensate for tissue scattering, the power to activate a cell at a specific depth z below the brain surface must be increased by a factor: = where Δ = ! − " . The maximum number of activable cells can be then estimated as +#7 ≈ √ for conventional or cyclic illumination, respectively.
For parallel illumination and high power (>10 W) low repetition rate lasers, the typical power for in vivo activation is in the range of 3-10 mW/cell [5][6][7][8] . Given these values and the total laser available power out of the objective, it is possible to estimate, for conventional and cyclic illumination, the maximum number of cells activable across a given depth in mouse brain.
We will here compare cyclic-FLiT and conventional holography for different experimental situations.
To facilitate the comparison, we will use a simplified 2-states model (as described in equation 4 and 11 of Supplementary Note 1). In this approximation, it is possible to describe the light-evoked photocurrent ( ) under a continuous power P of duration @AA as All these considerations will hold also at saturation, i.e. for ′ ≥ .#/ , however in this case both configurations (conventional holography and cyclic-FLiT) will give rise to a deterioration of the axial resolution.

Configuration 2)
Conventional holography and cyclic-illumination have different excitation powers and dwell times, and the two illumination protocols have the same total time of experiment (Supplementary Figure 21d): )WX ! By imposing that cyclic illumination reaches the photocurrents achievable with a constant illumination for a time t YZ IJI , one can write: For typically used value of Pstd and Pcyc and typical τ ]^^ values, one can assume and and a

result in agreement with what experimentally demonstrated in the manuscript (and derived in
Supplementary Note 1).
The requirement of using P IJI = √H. P [\Y will decrease the gain H in the achievable number of cells, found in configuration 1) by √H, so that the net gain in the achievable number of cells with cyclic-FLiT in this configuration is of √H times.
Similar to the discussion made above, for conventional holography reaching √H times more cells would require sequentially projecting √H holograms (each exciting N cells). This would increase the total illumination time to (texp+tSLM).√H and, importantly, will introduce a shift delay DT between the spiking time of the first and last cell (or group of cell) of (texp +tSLM)·(√H − 1). For the numbers demonstrate in our manuscript ( G7H = 5-10 ms and H ≤ 25), this would reach tens of milliseconds.
This is opposite to the case of FliT where this will be, as in configuration 1), equal to (H-1)·tcyc (so for the numbers demonstrated in our manuscript ≤ 1.2 ms).
The parameters characterizing the configuration 2 are summarized in Supplementary Table 2.

SUPPLEMENTARY TABLES:
Supplementary  Same as movie 1 but for FOV 260 x 260 µm 2 and time per frame 0.5 ms.